Mamoru Mimuro

EXCITATION ENERGY TRANSFER IN THE LIGHT HARVESTING ANTENNA SYSTEM OF THE RED ALGA Porphyridium cruentum AND THE BLUE‐GREEN ALGA Anacystis nidulans: ANALYSIS OF TIME‐RESOLVED FLUORESCENCE SPECTRA

Abstract— Time‐resolved fluorescence spectra of intact cells of red and blue‐green algae Porphyridium cruentum and Anacystis nidulans were measured by means of a ps laser and a time‐correlated photon counting system. Fluorescence spectra were observed successively from various pigments in the light harvesting system in the order of phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC) and chlorophyll a (Chl a). The spectrum changes with time in the range of0–400 ps in P. cruentum and of0–1000 ps in A. nidulans. The time‐resolved spectra were analyzed into components to obtain the rise and decay curve of each fluorescence component. Overall time behaviors of the sequential fluorescence emissions from various pigments can be interpreted with a decay kinetics ofexp(–2kt½). The rate constants of the energy transfer show that the energy transfer takes place much faster in the red alga P. cruentum than in the blue‐green alga A. nidulans, particularly in the step PCAPC. Results also indicated that a special form of APC, far‐emitting APC, exists in the pigment system of A. nidulans, but it does not mediate a main energy transfer from phycobilisome to Chl a.

SPECTRAL FORMS AND ORIENTATION OF BACTERIOCHLOROPHYLLS c AND α IN CHLOROSOMES OF THE GREEN PHOTOSYNTHETIC BACTERIUM Chloroflexus aurantiacus

Spectral forms of bacteriochlorophyll (Bchl) in chlorosomes were analyzed by linear dichroism, circular dichroism (CD), and deconvolution of these spectra. Isolated chlorosomes were embedded in polyacrylamide gels and compressed unidirectionally (along the x‐axis) while allowing the gel to stretch in another direction (along the z‐axis). The chlorosomes were aligned three‐dimensionally due to their flat oblong shape; the longest axis was presumed to parallel the z‐axis, its shortest axis was presumed to parallel the x‐axis, and the intermediate‐length axis was presumed to parallel the y‐axis. Degrees of polarization (AI− A1)/(AI+ A1) of Bchl c and a measured from the y‐axis with linearly polarized light were significantly different from those measured from the x‐axis. Deconvolution of spectra into components revealed the presence of two major forms of Bchl c with peaks at 744 nm and 727 nm. The degrees of polarization of the 744 and 727 nm spectral forms were 0.76 and 0.59 from the y‐axis and 0.48 and 0.39 from the x‐axis, respectively. The degrees of polarization of Bchl a794 were –0.21 from the y‐axis and 0.12 from the x‐axis. These values indicate that the direction of the Qy transition moment of Bchl c744 is almost completely parallel to the longest axis of chlorosomes and that of Bchl c727 is also nearly, but slightly less so, parallel to the longest axis of the chlorosomes. The Qy transition moment of the baseplate Bchl a peak at 794 nm is nearly perpendicular to the longest axis and parallel to the shortest axis: that is, it is perpendicular to the associated membrane plane in the cell. These alignments of Bchl transition moments in chlorosomes were lost after suspending the chlorosomes in a solution saturated with 1‐hexanol accompanying a shift in the peak position from 742 nm to 670 nm. The alignment recovered after the hexanol concentration was decreased. The presence of two major spectral forms of Bchl c was supported by the deconvolution of CD spectra and absorption spectra.

Carotenoids in photosynthesis: absorption, transfer and dissipation of light energy

The functioning of carotenoids in photosynthesis is discussed in relation to the reaction mechanism. The energy transfer process from the allenic carotenoid, fucoxanthin, to chl a was intensively investigated in the newly isolated fucoxanthin-chl a/c protein assembly (FCPA) from a brown alga Dictyota dichotoma. The transfer time was shorter than 3 ps at 15°C. The energy level responsible for transfer may not be the Qx band of chl a, contrary to the proposal for the transfer in the bacterial antenna system.

Excitation energy transfer in carotenoid-chlorophyll protein complexes probed by femtosecond fluorescence decays

Abstract An energy transfer pathway in a carotenoid-chlorophyll a protein complex of dinoflagellates was studied by the femtosecond up-conversion method. The energy levels of the S 2 state of peridinin and their lifetime were essentially identical in methanol and in the complex. The S 1 lifetime of peridinin in solvents was more than 30-fold longer than in the complex. These results account for an observed transfer efficiency (higher than 85%) and indicate that an energy transfer occurs between the S 1 states of peridinin and chlorophyll a after a rapid internal conversion. This pathway is unique in photosynthetic organisms.

Light-harvesting particles isolated from a brown alga, Dictyota dichotoma. A supramolecular assembly of fucoxanthin-chlorophyll-protein complexes

Using a new type of detergent, decylsucrose, light-harvesting pigment proteins of a brown alga, Dictyota dichotoma , were isolated as brown-colored particles (17.1 S). They are supramolecular assemblies consisting of 7 units of 4.8 S fucoxanthin-chlorophyll a/c protein complexes, but theirmolecular association is rather unstable, and they are readily dissociated to the 4.8 S complexes. The 4.8 S complexes have a very high pigment content (0.37 mg/mg protein); 10 fucoxanthin, 1 violaxanthin, 3 chlorophyll c and 13 chlorophyll a being confined in a 54 kDa protein to form a 4.8 S pigment-protein (molecular weight 74 · 10 3 ). In the intact assemblies, excitation energy migrates through two separate paths, from fucoxanthin to chlorophyll a and from chlorophyll c to chlorophyll a , both with the efficiencies as high as from chlorophyll a itself. Fucoxanthin and chlorophyll a are assumed to form donor-acceptor couples at a 1:1 ratio, and this would probably be the case for chlorophyll c and chlorophyll a . On contact with Triton X-100 (0.01%), these particles were dissociated into component proteins with a concomitant blue-shift of fucoxanthin, and consequent breakdown of the coupling between fucoxanthin and chlorophyll a .

Molecular structure and optical properties of carotenoids for the in vivo energy transfer function in the algal photosynthetic pigment system

Common fluorescence properties of carotenoids functioning as an efficient antenna in algal pigment systems are elucidated in relation to their molecular structure. Those carotenoids contain eight conjugated double bonds and one keto group associated with the double bond. The origin of the fluorescence is the optically forbidden S 1 state and its radiative lifetime is longer than that of carotenoids without a keto group. These characteristics are discussed in relation to the excited state of polyenes.

Dynamic fluorescence properties of D1-D2-cytochrome b-559 complex isolated from spinach chloroplasts: Analysis by means of the time-resolved fluoresence spectra in picosecond time range

Abstract Dynamic fluorescence properties of D1-D2-cytochrome b -559 complex isolated from spinach chloroplasts were analyzed with time-resolved fluorescence spectra and deconvolution of the spectra. The changes in the spectra at 4° C were fitted by the changes in the relative contribution of at least three fluorescence components whose emissions peak at 670, 676 and 682 nm (F 670 , F 676 and F 682 ). The F 670 and F 676 are interpreted to arise from accessory chlorophyll a and the F 682 from the primary donor of reaction center II (P-680), based on the kinetic response and the energy level. The decay kinetics of F 682 is composed of two components of lifetimes about 25 ps and 35 ns. The former lifetime corresponds probably to the kinetics of charge separation while the latter, to the kinetics of charge recombination between P-680 and pheophytin. The spectra at −196° C were fitted by four components, including the above three and an additional band peaking at 693 nm (F 693 ). The F 683 , which corresponds to the F 682 at 4° C, was the main band throughout the measuring time up to about 80 ns. Its decay kinetics had two components with its slow phase of about 40 ns. This corresponds to the time for the charge recombination at the low temperature. The rate constant and the absolute yield of the F 683 slowest component (charge recombination) were almost independent of temperature, while the fluorescence yield of the other decay components was temperature dependent. This gave rise to the changes in the time-resolved fluorescence spectra under two temperature conditions.

Spatial arrangement of pigments and their interaction in the fucoxanthin-chlorophyll ac protein assembly (FCPA) isolated from the brown alga Dictyota dichotoma. Analysis by means of polarized spectroscopy

Molecular interaction and energy transfer between pigments in the fucoxanthin-chlorophyll (Chl) ac protein assembly (FCPA) isolated from the brown alga Dictyota dichotoma was investigated mainly by polarized spectroscopy. FCPA consists of 7 identical units of 54 kDa apoprotein, each of them containing 13 Chl a, 3 Chl c, 10 fucoxanthin and 1 violaxanthin. Spectral heterogeneity was found in the component pigments; two types of Chl a (Chl a673 and Chl a669), two types of Chl c (Chl cl, long-wavelength form of Chl c and Chl cs, short-wavelength form). In fucoxanthin, two functionally active and one inactive species were found. Energy flow in the FCPA is attained by a direct coupling of donor-acceptor pair and those are classified into four types: from fucoxanthin to Chl a669, from fucoxanthin to Chl a673, from Chl cl to Chl a669 and from Chl cs to Chl a669. The number of those four pathways was estimated to be 7, 2, 2 and 1, respectively, per unit peptide. Energy migration in the Chl a molecules is always functioning. Dissociation of FCPA into unit peptides induces the uncoupling of energy transfer between the respective donor and acceptor Chl a. The spatial orientation of individual pigments, investigated by linear dichroism and polarized fluorescence spectroscopy, was shown to be favorable for an efficient energy transfer. Based on the results of polarized spectroscopy, a spatial orientation of individual chromophores in the peptide was proposed.

Excitation energy transfer in phycobilisomes at −196°C isolated from the cyanobacterium Anabaena variabilis (M-3): evidence for the plural transfer pathways to the terminal emitters

Excitation energy transfer in phycobilisomes at −196°C was studied by means of the time-resolved fluorescence spectroscopy in the picosecond time-range. Supplemental data were obtained from the allophycocyanin-core complex. When the phycobilisomes were excited at 580 nm, at leastnine fluorescence components were resolved by the time-resolved spectra and deconvolution of thosespectra. Energy transfer among these components is not straightforward. At the phycocyanin level, two transfer pathways are probable; one may be among the β-155 chromophores, and another among α-84 chromophores along the long axis of the phycobilisome rods. The β-84 chromophores in intermediate discs of the rods might function as an energy pool by a fast equilibrium between αa-84 and β-84 chromophores. The β-84 chromophores in the trimer next to the core-complex is the energy donor to the core-complex. At allophycocyanin level, two pathways of the energy transfer were also found; one from F660 to F686 through the F673. This corresponds to the energy flow from β-84 chromophore without linker to 18.3 kDa polypeptide and finally to the ‘anchor’ polypeptide. The other pathway is from the F666 to F680, i.e., from the β-84 with a linker polypeptide to the α-subunit of allophycocyanin B. Two independent pathways in the energy transfer shown in this study basically agreewith the assembly model of the core components proposed by Glazer (cf. Biochim. Biophys. Acta 768 (1984) 29–51).

Topology of pigments in the isolated Photosystem II reaction center studied by selective extraction

Abstract Pigments in the purified spinach Photosystem II reaction center (D1-D2-Cyt b-559) complex were extracted with diethyl ether containing varied amounts of water. The purified reaction center originally contained approximately six molecules of chlorophyll a, two β-carotene and two pheophytin a per one photochemically active pheophytin a. The treatment with 30–50% water-saturated ether extracted one β-carotene, as well as one chlorophyll a that absorbs at 677 nm, remaining 62% of the photochemical activity to reduce pheophytin a. With 60–80% water-saturated ether, almost all the β-carotenes were extracted, remaining the 49% activity without additional loss of chlorophyll. The absorption, fluorescence excitation and linear dichroism spectra demonstrated two spectral forms of β-carotene. The short-wavelength form of β-carotene with absorption peaks at 429, 458 and 489 nm was selectively extracted with ether at low water content, whereas the long-wavelength form with peaks at 443, 473 and 507 nm was extractable only at the higher water content. The extraction enhanced the photobleaching of chlorophylls. The results suggest that chlorophyll a forms with peaks at 667 and 675 nm are located close to the short-wavelength form of β-carotene that can transfer excitation energy to the photoactive pheophytin a on the D1 protein.